Process record slide for staining and method of using the same

ABSTRACT

Described herein is a process record slide for staining. The process record slide includes a detection area for mounting a sample and a control area including one or more control targets. Also described herein is a method of using the process record slide in an immunohistochemical (IHC) or an immunochemistry (ICC) staining process.

PRIORITY CLAIM AND CROSS-REFERENCE

The instant application claims priority from U.S. ProvisionalApplication No. 62/745,074, filed Oct. 12, 2018, the entirety of whichis hereby incorporated herein by reference.

BACKGROUND

Immunohistochemistry methods as well as other immunochemical methods aremulti-step procedures. These methods include a sequence of reagentexchanges, incubations, and washings. The procedures require skilledpersonnel and often the use of an automated staining machine. Resultscan vary significantly among laboratories or institutions because ofnon-uniformity, or lack of, in process quality control standards.Diagnostic interpretation of the processed slide relies on subjectiveanalysis by a pathologist, who in most cases was not involved with thestaining process.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description when read with the accompanying figures. It isnoted that, in accordance with the standard practice in the industry,various features are not drawn to scale. In fact, the dimensions of thevarious features may be arbitrarily increased or reduced for clarity ofdiscussion.

FIG. 1 is a process record slide in accordance with some embodiments.

FIG. 2 is a process record slide in accordance with some embodiments.

FIG. 3A is a process record slide during the manufacturing process inaccordance with some embodiments. FIG. 3B is a process record slideafter the manufacturing process is finished in accordance with someembodiments. FIG. 3C is a process record slide after the stainingprocess in accordance with some embodiments.

FIG. 4 is a process record slide including a paraffin coating inaccordance with some embodiments.

FIG. 5A and FIG. 5B are a primary target density gradient array and asecondary target density array of the slide in accordance with someembodiments.

FIG. 6 is an antigen retrieval target of the slide in accordance withsome embodiments.

FIG. 7 is a control area of the slide in accordance with someembodiments.

FIGS. 8A and 8B are slides after the staining process in accordance withsome embodiments.

FIG. 9 is a staining process using a slide in accordance with someembodiments.

FIG. 10 is an imaging result of a stained slide in accordance with someembodiments.

FIG. 11 is an enzyme amplification process in accordance with someembodiments.

FIG. 12 is a single primary antibody control slide that works with anymouse or rabbit based primary antibody in accordance with someembodiments.

FIG. 13 is a dual primary antibody control slide that supports the useof one mouse based primary antibody and one rabbit based primaryantibody in accordance with some embodiments.

DETAILED DESCRIPTION

The following disclosure provides many different embodiments, orexamples, for implementing different features of the provided subjectmatter. Specific examples of components and arrangements are describedbelow to simplify the present disclosure. These are, of course, merelyexamples and are not intended to be limiting. For example, the formationof a first feature over or on a second feature in the description thatfollows may include embodiments in which the first and second featuresare formed in direct contact, and may also include embodiments in whichadditional features may be formed between the first and second features,such that the first and second features may not be in direct contact. Inaddition, the present disclosure may repeat reference numerals and/orletters in the various examples. This repetition is for the purpose ofsimplicity and clarity and does not in itself dictate a relationshipbetween the various embodiments and/or configurations discussed.

For immunohistochemistry methods and other immunochemical methods, bothautomated and manual procedures include steps where care should beexcised. For example, care must be exercised to avoid the loss of or todamage the specimen on the slide. Since thorough washing of the specimenbetween reagent applications is used to remove any unbound antibody, asresidues would be amplified, and excess liquid is removed to avoidcarryover of the previous reagent and/or unwanted dilution of thesubsequent reagent, yet specimens cannot be allowed to dry out. Inaddition, sufficient antibody reagents must be applied to cover theslide area, but waste needs to be kept to a minimum as the reagents arecostly.

In addition, reagents used in immunohistochemical methods as well asimmunochemical methods, such as enzyme solutions and peroxidase colordevelopment reagents, have limited stability at working temperature orat room temperature. The limited stability results in frequentpreparation of the reagents. Furthermore, nonspecific antibody bindingwhich leads to erroneous results remains a problem.

Immunohistochemical (IHC) staining in general, is used to assess thepresence of specific antigen sites in a patient tissue section. In IHCassay, subjective interpretation is applied against the stain density onthe tissue section to assign the diagnostic level of an abnormal orcancerous condition. An assumption is often made that the IHC processingfunctions correctly and that the tissue section would be marked withvisible chromogen markers identifying the abnormal or cancerousconditions if the markers are present. However, failure of the antigenretrieval process or mistakes on the part of the lab technician orduring the stain processes often leaves no identifiable artifacts. Whenthis happens, the IHC assay provides nothing more than what would havebeen provided by a hematoxylin and eosin (H&E) staining. In addition,the failures or mistakes in the staining process sometimes leads tomisdiagnosis, which affects the subsequent treatment.

Methods and reagents that improve results and minimize reagentpreparation would facilitate both manual and automatedimmunohistochemical methods. Many of the improvements could be readilyapplied to related immunochemical methods such as enzyme-linkedimmunosorbent assays (ELISA), immunofluorescence assays and in situhybridization.

Due to the above, completely eliminating errors in theimmunohistochemistry methods and immunochemical methods is difficult. Assuch, various methods of revealing the existence and/or extent of theerrors have been developed. Some of the methods as well as thelimitations of the methods will be described below.

Reference may be made to “Use of cultured cells as a control forquantitative immunocytochemical analysis of estrogen receptor in breastcancer the Quicgel method” (the entirety of which is hereby incorporatedherein by reference). This references states that the variation intissue fixation, processing, and staining is largely responsible forpoor reproducibility of estrogen receptor (ER) immunohistochemistryassays. A frozen, agar-suspended pellet of MCF-7 cells with known ERcontent was added to each of 55 samples of invasive breast carcinoma(IBC), serving as a control. Image analysis determined percentages ofpositive area (positive nuclei per total nuclei analyzed) and positivestain (sum of optical density of the positive nuclear area divided bysum of the optical density of all nuclei studied) of MCF-7 cells andIBC. MCF-7 cells had a mean value of 150 fmol/mg of ER by dextran-coatedcharcoal analysis. Image analysis of MCF-7 cells included with the 55cases showed a mean positive area of 70.81. Positive staining from theIBC cases ranged from 0 to 98.5. By using the known ER content and thepositive area of the MCF-7 cells, a conversion factor was used totranslate the positive area of the clinical specimens to a femtomoleequivalent, which for the 55 IBCs ranged from 0 to 1,790 (mean, 187).Inclusion of a control with known femtomole quantity of ER provides aninternal standard for quality control and ER quantitation.

Reference may be made to CN102435728 (the entirety of which is herebyincorporated herein by reference). This reference describes a method ofpreparing and utilizing a positive control for inspecting andcontrolling the quality of immunohistochemical processes. According toCN102435728, a control target is constructed by adsorbing a polypeptideor a protein having specific reaction with an antibody on a slide atdifferent concentrations. A sample tissue is then mounted on the slideand a conventional immunohistochemical process is performed on theslide. Because the sample tissue and the control targets undergo thesame staining process, the staining result of the polypeptide/protein isindicative of the results of the immunohistochemical process on thetissue. CN102435728 also describes arranging the positive controlprotein or polypeptide on the immunohistochemical slide.

One of ordinary skill evaluating the method of CN102435728 wouldunderstand that the density of the protein/peptide in the control targetis inconsistent. The reason is that binding of the peptide segments tothe dextran polymer depends on viscosity of the mixing solution, washingprocess that removes excess peptides from the dextran, and temperature.In addition, the size of the precipitated polymer pellets varies withthe bath concentration, the reaction temperature, and the NaOHinjection.

One of ordinary skill evaluating the method of CN102435728 wouldunderstand that the building targets of known reactivity (stain density)is difficult. The reason is that the concentration of available peptidesin the polymer pellets cannot be easily derived. As such, the resultedcontrol is only a yes/no primary antibody detector.

One of ordinary skill evaluating the method of CN102435728 wouldunderstand that while the dextran is able to capture proteins forantibodies to target, the captured protein only provides a yes/noresult. Thus, a baseline detection ruler cannot be established tosupport, e.g., a digital imaging.

One of ordinary skill evaluating the method of CN102435728 wouldunderstand that the proteins/peptides absorbed on the slide often leakduring the antigen retrieval process. Due to the relatively small sizeof the slide and therefore the close proximity between the controltargets and the sample area, the leaked proteins/peptides can move tothe tissue section. As such, the leakage can bind to the co-residenttissue section and slide adhesive to cause non-specific staining.

Reference is made to Horizon Diagnostics, which provides slidesconstructed differently from those as described in CN102435728. In theslides of Horizon Diagnostics, the control targets are cultured celllines. The cell lines are genetically engineered to include codingsequences of specific antigen peptides in the DNA. The geneticallyengineered cells are fixed in tissue blocks in the slide by formalin andparaffinized in the form of a loose cell slurry. In addition, controltargets including non-reactive cells are also included in the slides asnegative controls. To produce an array of control targets, each cellgroup are formed as a cylinder core aligned with other cores, and theentire array is cut as a single section for application to the slide.These cell lines can be replicated as desired and therefore consideredas renewable.

One of ordinary skill evaluating the slides made by Horizon Diagnosticswould understand that the control targets can only provide a yes/noresult due to the uncontrollable density variations of the reactiveantigens presented in the control targets. The reason is thatcontrolling the amount of cells or the amount of antigens produced bythe cells are difficult. As such, each control target includes differentamounts of antigens. In addition, since the control targets go throughthe antigen retrieval process, which introduces another variable to theantigen amount in the control targets.

One of ordinary skill evaluating the slides made by Horizon Diagnosticswould understand that the amount of antigen in each cell well cannot becontrolled by simply mixing an antigen producing cell line and a blankcell line in certain ratios. The reason is that the electrostatic chargeon the different cell lines are often different. As such, the differentcell lines often dissociate during the cell culture process and growinto sections of different cells, making the generation of antigendensity scale improbable or even impossible.

One of ordinary skill evaluating the slides made by Horizon Diagnosticswould understand that there is inconsistent cell line performance sincethe cell reproduction has a limited replication life. There can be noassurances that a new cell line will have the same antigen density asany previous cell line.

One of ordinary skill evaluating the slides made by Horizon Diagnosticswould understand that making the control target of Horizon Diagnosticsis not cost effective due to the manual labor required to constructtissue blocks, section the blocks, and apply cut section to the slide.

Reference may be made to US2016/0274006A1 (the entirety of which ishereby incorporated herein by reference) which describes method andapparatus that serve as a control and calibrator for assays performed oncells and tissues mounted on a microscope slide. The apparatus ofUS2016/0274006A1 includes a quality control moiety, such as a peptideepitope, linked to a particulate object, such as a clear spherical beadand the bead is preferably approximately the size of a cell. The qualitycontrol moiety is designed to behave in a similar manner in the assay asan analyte, yielding a positive assay reaction. The bead is retained ona microscope slide during the steps of staining by a novel liquidmatrix, which solidifies upon drying and causes adherence of the beadsto the microscope slide.

This control and calibrator solution of US2016/0274006A1, however, haslimited practicality. Because that the control target material is coatedon beads and sparsely located, the stability of the targets tends to beweak and the target data is difficult to extract. In addition, whenimaging a single bead, the stain color changes from the top center tothe rim, thus introducing additional variables.

Reference may be made to US7271008B2 (the entirety of which is herebyincorporated herein by reference) which describes a device and methodsfor determining the quality of reagents used in an assay process,particularly a multistep immunohistochemical assay. In particular, thedevice comprises a substrate with a plurality of compounds affixed to asubstrate, where each compound is reactive with a reagent used in theassay.

The device of US7271008B2 provides a quality control that evaluates thestaining by the secondary antibody. However, the device of US7271008B2does not undergo the same steps of an IHC staining together with thesample. Therefore, representativeness of the IHC staining by the deviceis limited. In addition, the device uses amino-silane in the substrate,which could not form covalent bonds that are capable of surviving theantigen retrieval processing. Also, the alkaline phosphatase targetwould breakdown upon exposure to the antigen retrieval temperatures. Assuch, the device of U.S. Pat. No. 7,271,008B2 is not useful inmonitoring the antigen retrieval process, which is known to introduceunknown variables in the IHC staining process.

In view of the above, the instant specification, in some embodiments,describes a process record slide and a method for staining thataddresses these issues. Specifically, the instant specificationdescribes a process record slide that allows a patient sample andcontrol targets to co-reside and experience the staining processtogether. As such, in the processing of the sample, the control targetsare also stained, which reveals the existence and extent of processingerror(s) as deviation against a known target baseline. One of ordinaryskill in the art would recognize that changes in concentration of thestaining reagents applied to a sample will impact subjective analysis.Excessive primary antibody often causes non-specific staining on thetissue section. Ignoring the antigen retrieval processing, the efficacyof the primary antibody, secondary antibody, and chromogen reagents willaffect the stain color density (the indication of antigen density on thetissue section.) Aging of antibody proteins results in the hinge betweenthe Fab and Fc domains breaking soon followed by splitting of the Fcdomain. Only those antibodies containing both Fab and Fc domains willresult in successful operation. Many of the chromogen reagents,containing the colorant for precipitation, have short lifetimebehaviors, some as short as hours. As a result, chromogen reagents ofdifferent ages will have different performances leading to variations instain color density. Thus, the performance of the reagents can and doimpact the stain color density in the observed sample. Since the samestain reagents are used for both the sample and the control targets,variation in reagent age and amount is accounted for by the controltargets in order to provide more objective information for analysis.According to these embodiments, the process record slides are able todeliver an effective Process QC with desirable accuracy and precisionwithin a cost sensitive price point such that it can be used on everyIHC and ICC slide.

Process Record Slide

In some embodiments, the instant specification is directed to a processrecord slide. In the instant specification, the term “process recordslide” is alternatively referred to as a “slide”, a “PRS” or a“PRS-IHC.” The “IHC” portion in the term “PRS-IHC” is not meant to limitthe construction or the function of the slide to immunohistochemcial(IHC) staining only. Rather, one of ordinary skill in the art wouldunderstand that the process record slide as described herein can be usedin other staining processes, such as immunochemistry (ICC), as well.

In some embodiments, the instant specification is directed to a processrecord slide that allows easy determination of the efficacy of stepscommonly involved in immunohistochemcial (IHC) or immunochemistry (ICC)assays. These steps include paraffin removal, antigen retrieval, primarystaining, secondary staining and etc.

Refer to FIG. 1, in some embodiments, the process record slide 100includes a substrate 101, an adhesive layer 103, and one or more controltargets 141.

In some embodiments the substrate 101 includes a glass substrate, aplastic substrate or a polymer substrate.

In some embodiments, the adhesive layer 103 includes an adhesive. Insome embodiments, the adhesive binds covalently to the substrate 101,such as a glass substrate.

In some embodiments, the adhesive provides a slightly hydrophilicsurface on the slide. In some embodiments, a material of the adhesiveincludes an end group that is presented to biomaterials and isadjustable at the time of manufacturing for the surface wettability. Insome embodiments, the end group includes one or more groups selectedfrom the group consisting of —ROH, —R(C═O)OH, —RNH₃, —R(C═O)NH₂, and—RNH₂. In some embodiments, the adhesive layer 103 includes an adhesivethat is the same as or similar to the adhesive found in the coatings ofThermo-Fisher SuperFrost slide GL4951P.

In some embodiments, the one or more control targets 141 are depositedon the substrate layer via the adhesive layer. In some embodiments, theone or more control targets are deposited in the control area 140 of theslide 100.

In some embodiments, the control targets 141 includes chemical compoundsthat react with one or more reagents used in the IHC or ICC process andproduce a reading, such as a reading in the form of a color. Each of thecontrol targets 141 includes a different concentration of chemicalcompounds. The variation in the concentration in the chemical compoundsprovides an identifiable difference between the control targets 141following interaction with the one or more reagents in the IHC or ICCprocess.

In some embodiments, the process record slide 100 further includes aprotective coating 105 covering an entirety or a portion of the slide100. In some embodiments, the protective coating 105 seals the controltargets 141 from the outside environment. As detailed below, the controltargets 141 sometimes include proteins, peptides or other targets ofbiological origin. Therefore, the protective coating 105 protects thecontrol targets 141 from oxidation or microbial attack.

In some embodiments, the protective coating 105 is a paraffin coating.According to these embodiments, the paraffin coating is removed togetherwith the embedding paraffin on the tissue/cell sample to be studied bythe IHC or ICC assay by the same de-paraffinization step.

Refer to FIGS. 1-3C, in some embodiments, the process record slide 100includes a detection area 120 configured to hold a sample, and a controlarea 140.

In some embodiments, the detection area 120 is configured to hold apatient sample, such as a tissue section, or loose cells.

In some embodiments, the control area 140 holds one or more controltargets 141 that undergo the staining process together with the sampleheld in the detection area.

In some embodiments, the detection area 120 and control area 140 have amarked boundary. In some embodiments, the detection area 120 and controlarea 140 do not have a marked boundary and are classified based on therespective function.

In some embodiments, the control targets 141 are arranged in the form ofone or more control target loading dots 1411. In some embodiments, theloading dots are of a regular or an irregular shape. In someembodiments, the regular shapes including circles, ellipses, squares, ordiamond shapes. In some embodiments, the control targets 141 arearranged in a 2D or a 3D configuration.

In some embodiments, the control targets 141 are arranged in the form ofone or more control target arrays 1415, such as one or more arrays ofcontrol target loading dots 1411. In some embodiments, each of thecontrol target arrays 1415 include one type of control targets that havethe same or similar chemical and biochemical properties. In someembodiments, each of the control target arrays 1415 include one type ofcontrol targets that result in contrasting optical properties.

According to these embodiments, because the detection area 120 and thecontrol area 140 are located on the same slide, during the IHC or ICCprocess, the samples held in the detection area 120 and the controltargets 141 in the control area 140 undergoes the same staining steps(refer to FIG. 9). As such, the level of staining of the control targets141 reveals the existence and extent of processing errors occurredduring the IHC or ICC process, and the control targets 141 allow theprocessing errors of the IHC or ICC process to be analyzed as deviationagainst a known target baseline. Therefore, according to theseembodiments, the process record slides are able to deliver an effectivequality control with desirable accuracy and precision, with reducedsubjective analysis, at a relatively low cost, resulting with an easyacceptance threshold. Furthermore, with subsequent digital image captureand processing, the stained control targets 141 can serve as an antigendensity ruler, containing two scales: numerical antigen density andcolor density which can be applied to the co-resident sample to provideaid in, e.g., diagnostic determination.

In some embodiments, the control targets 141 are deposited on the slidein such manners that the control targets 141 are not significantlyaffected by the pretreatment steps commonly used in IHC or ICC staining.In some embodiments, the pretreatment steps include de-paraffinizationor antigen retrieval. The pretreatment steps is described in below indetail.

In some embodiments, the slide 100 further includes a slide informationarea 160. In some embodiments, the slide information area 160 includesinformation on the type of the slide such as a lot number, date ofmanufacture, control target types, etc. Since the slide information,such as the information on control target types, is extensive, in someembodiments, the slide information area 160 includes a barcode recordingthe slide information. In some embodiments, the barcode includes awebsite link to a detailed description of the slide.

Refer to FIG. 3, in some embodiments, the slide 100 further includes abulging structure 107. According to these embodiments, the bulgingstructure 107 ensure that when stacked, the adjacent slide does notdamage the paraffin coating or the control targets. In some embodiments,the bulging structure 107 includes a pair of bars or multiple dotsextending beyond the control area 140. These bumpers help to the directprint ink-jet label printers commonly feed unmarked slides from thebottom of the magazine. Thus, the bumpers ensure that the paraffincoating and targets below are undamaged when one slide slides overanother during the dispensing from the magazine.

Protective Coating

In some embodiments, the protective coating 105 is a paraffin coating.

Refer to FIG. 4, in some embodiments, the paraffin coating is a coatingof paraffin wax on the control targets that seals the control targets.

Paraffin wax, in general, is a white or colorless soft solid, derivedfrom petroleum, coal or oil shale, which consists of a mixture ofhydrocarbon molecules containing between twenty and forty carbon atoms.It is solid at room temperature and begins to melt above approximately37° C. (99° F.); its boiling point is >370° C. (698° F.). Commonapplications for paraffin wax include lubrication, electricalinsulation, and candles; dyed paraffin wax can be made into crayons. Itis distinct from kerosene and other petroleum products that aresometimes called paraffin.

In a pathology laboratory, water free paraffin wax is used to impregnateformaldehyde fixed tissue prior to sectioning thin samples of tissue. Inthis procedure, water is removed from the tissue through ascendingstrengths of alcohol (75% to absolute) and the tissue is cleared in anorganic solvent such as xylene or one of the aliphatic substitutes, suchas Xylol. The tissue is then placed in liquid paraffin wax for a periodtime in a vacuum oven to ensure that all air is extracted and then setin a mold block frame with liquid wax to cool and solidify. The sectionsare then cut on a microtome.

Embedding tissue sections into paraffin is a practice for thepreservation of the biopsy tissues for a prolonged period of time.However, to the best knowledge of the instant inventors, the applicationof paraffin as a coating layer on a selected area of a microscope slidehas not been reported.

As described below, control targets slide includes peptides or proteins,and therefore present a rich food source for bacteria or fungi. Thepeptides and proteins, as well as the antigen sites thereof (e.g.epitopes) are susceptible to oxidation which sometimes compromises theability to bind antibodies. In addition, of the subsequent reactioninvolves hydroxyl groups, which can be damaged through reactions withairborne acids and bases. As such, slides containing protein depositsare often required to be stored at temperatures below what supportsmicrobial growth, and need to be packaged in vacuum sealed. Unprotectedslides, such as those exposed to environment, have shelf lives as shortas 2 to 5 days depending upon ambient temperature, humidity, andairborne contaminate levels. Such are a constraint that limits theeffective utilization of deposits.

Paraffin has anti-fungal and antibacterial capability and preventsoxidation of materials sealed under. The instant inventor has found thatthe paraffin coating extends viable life of the biomaterials from 3-5days to 1-2 years, thereby significantly increases the shelf life of theprocess record slide.

Removal of the embedding paraffin from the sample is commonly performedduring IHC or ICC staining (this process is referred to as“de-paraffinization”). Therefore, in some embodiments, a formulation ofthe paraffin included in the protective layer 105 is the same as orsimilar to the formulation of the paraffin used in the embeddingprocess. As such, the paraffin coating can be removed at the same timeof the de-paraffinization, thereby simplifying the staining process.

In some embodiments, paraffin is liquefied, applied on the slide andsolidified, thereby forming the paraffin coating.

In some embodiments, the paraffin is blended with a solvent to changethe material state from solid to liquid at room temperatures. In someembodiments, the solvent includes, Xylene or an Aliphatic solvent, forexample Xylol. The blending with the solvent reduces the viscosity andslows down solidification following deposition. In some embodiments, thesolvent includes toluene, paint thinner, turpentine, or a 50:50 mix ofacetone & kerosene. Paraplast X-tra additionally includes butylatedhydroxytoluene, a phenolic antioxidant, further reducing the oxidationdegradation of protein, peptide, or inorganic targets.

In some embodiments, the solid paraffin is melted at a temperature of nomore than 75° C. above the paraffin melt temperature. The meltedparaffin is then slowly added with an aliphatic solvent until thesaturation point is observed (i.e., solids are formed). The mixture isallowed to cool to about 45° C. Additional aliphatic solvent is thenadded until the mixture is completely clear.

In some embodiments, the paraffin coating is applied over biomaterialand special stain reactive deposits previously deposited to a microscopeslide. The biomaterials, as described below, include proteins, peptides,conjugated proteins, protein coated beads, peptide coated beads,conjugated coated beads, special stain reactive end groups that uniquelycapture a stain material, and etc.

In some embodiments, the paraffin is applied onto the slide by spraying,inkjet deposition, transfer printing such as pad printing, screenprinting, and vapor deposition. In some embodiments, the paraffincoating is applied onto the slide by heating to melt and/or blendparaffin particles into a monolithic surface coating sealing both thecontrol targets and a slide surface surrounding the control targets.

In some embodiments, after the paraffin coating is applied onto theslide, the slide is heated to drive off the solvent from the paraffin,thereby ensuring that the paraffin returns to a hardened state. Aprocess for removing the solvent is performed by infrared light appliedfrom the paraffin wax side of the slide. This method preferentiallytargets the solvent over the paraffin or biomaterials that are below theparaffin, thereby minimizing the needed thermal energy. The evaporatedsolvent is free to leave the paraffin without impediment.

In some embodiments, the paraffin is a blend of purified paraffin,synthetic polymers, and other materials. In some embodiments, desirablemelting temperature, hardness, and viscosity of the paraffin is obtainedby experimenting on ratios of the components. However, for the purposesof protecting biomaterials the paraffin is purified and water free.

In some embodiments, the paraffin coating has a thickness of 5 micronsor less.

In some embodiments, the paraffin in the protective coating 105 has amelting temperature of less than 60° C., such as less than 56° C.

In some embodiments, the paraffin is dissolvable by xylene, xylol, or analiphatic replacement thereof.

In some embodiments, the paraffin formulation includes, but limited to,TissuePrep & TissuePrep 2 by Thermo Fisher, melting temp 56° C.,Paraplast & Paraplast Plus by Leica, melting temp 56° C., and ParaplastX-tra by Leica, melting temp 50-54° C. In some embodiments the ambienttemperature hardness of the paraffin coating is chosen to be the hardestformulation available with butylated hydroxytoluene added if not alreadyin the formulation.

Primary Targets and Primary Target Arrays

In some embodiments, the control targets 141 include one or more primarytargets to test primary antibody reagents.

As used herein, the term “primary targets” means a composition ofreactive and non-reactive elements that specifically bind to a primaryantibody by only one of its FcyR1 sites. As used herein, the term“primary protein” means a protein that binds to the FcyRI of the primaryantibody. As used herein, the term “primary dummy protein” means aprotein that will not react to any primary antibody or secondaryantibodies. As used herein, the term “maximum target density” refers tothe maximum monolayer density of adjacent proteins. As used herein, theterm “surrogate antigen” refers to a protein that is not targeted by theprimary antibody used in the IHC or the ICC assay, but nonetheless bindsto the primary antibody via, for example, only one of the two FcyRIregions of the primary antibody (mouse or rabbit based) used in the IHCor the ICC assay. As used herein, the term “mouse only surrogateantigen” means a protein or antigen that uniquely reacts to only to theFcyRI of mouse based primary antibody. As used herein, the term “rabbitonly surrogate antigen” means a peptide or protein that uniquely reactsto only the FcyR1 of a rabbit based primary antibody.

Placenta mammal proteins range between 50-65 kDa in weight. Primaryantibody IgG proteins are composed of two Fab and one Fc domains thatare usually modeled with a Y-shape. The Fc is joined to the two Fabdomains by a hinge. All non-conjugated antibodies are grown within ahost mammal (most often Mouse or Rabbit) through inoculation of the hostby an antagonist antigen peptide. The host produces antibodies to fightoff the offending antagonist antigen peptide by assembling a bindingamino acid sequence near the N-terminal end of the antibody's Fab domainthat can capture the mating antigen. The Fc domain of the antibody isalways that of the host mammal. The Fc domain of the host antibodytypically has three binding sites available to the secondary antibody inrank order of highest binding affinity to lowest: FcyRI (CD64), FcyRII(CD32), and FcyRIII (CD16).

The primary dummy protein is chosen to be unreactive to mouse, rabbit,goat, bovine, and sheep as these can often bind to the secondaryantibody. In some embodiments, the dummy primary protein is any memberof the Equine family: horse, donkey, zebra, taper, or rhino. BSA (bovineserum albumin) is not a suitable protein as BSA will not bind covalentlyto the tissue or most slide adhesive chemistries without heat and time.Bovine, sheep, and goat are also not suitable dummy proteins as in somecases these proteins may be confused with mouse or rabbit depending onthe specificity of the primary antibody.

The primary protein is either host anti-mouse or host anti-rabbit. Thehost is limited to goat or any member of the equine family.

Primary proteins based on peptides can also be composed of mouse orrabbit FcyRI reactive peptides with cysteine residues at the C-terminalend covalently attached to a carrier protein such as KLH subunit(keyhole limpet hemocyanin). The KLH subunit is activated withSulfo-SMCC at its free amine sites, which through conjugation binds tothe cysteine residue of the peptide. Un-activated KLH subunit alonesuffices to make the primary dummy protein.

It is important to note that KLH as a full protein is approximately 8000kDa. In the full protein state, it is not stable in regards to pH ortemperature, which will cause the protein to separate into subunits:KLH1 at 390 kDa and KLH2 @ 350 kDa. Either of these subunits can be usedas carrier proteins, when Sulfo-SMCC activated, for peptides with acysteine residue, usually at the C-terminal end of the peptide.

Refer to FIG. 5A, in some embodiments, the primary targets are arrangedinto a primary target array 1415. In some embodiments, the primarytarget array 1415 is a primary target density gradient array. Eachtarget within the array must have the same maximum target density(proteins/surface area). However, the ratio between primary and dummyprimary proteins are stepped in ratio to ensure that a wide range ofprimary antibody dilutions can be identified. As a general rule,detection by two primary surrogate antigen targets is sufficient to mapthe concentration to the detection transfer curve. In some embodiments,the primary target density gradient array includes a plurality ofprimary target loading dots wherein one array is mouse based and theother rabbit based.

In some embodiments, the density of the primary targets increases ordecreases in a linear manner, such as 100%, 80%, 60%, 40%, etc. In someembodiments, the density of the primary targets increases or decreasesin a logarithm manner, such as 100%, 33%, 10%, 3%, etc.

In some embodiments, in a primary target density gradient array, in theloading dot having the highest primary target density, the primarytarget density is sufficiently high so that the primary target is notable to saturate the loading dot during the staining process.

In some embodiments, the universal surrogate antigen is either Protein Aor Protein G. Protein A binds to the FcyRI and to some areas within theFab domain of placenta mammals. Protein G will bind only to the FcyRIsite of most placenta mammals.

In some embodiments, the surrogate antigen must have specificity tomouse or rabbit only. To support the specificity, the surrogate antigencan be composed of anti-mouse and anti-rabbit proteins, anti-mouse andanti-rabbit peptides on a carrier protein, or anti-mouse and anti-rabbitVHH protein domains all respectively.

In some embodiments, the dummy surrogate antigen includes an antibodyfrom an animal of the equine family or a hoofed placenta mammal exceptgoat. Equine family includes animals such as horses, donkeys, tapirs,rhinos and mules. The equine family are evolutionarily and geneticallyfurther apart thus reducing the occurrence of non-specific staining.Goat cannot be used as often the secondary stain uses an anti-goatprotein in its sequencing, which would bind to any unreacted goatprimary sites.

Although the manner of interaction between the surrogate antigen and theprimary antibody used in the staining process is different from thosebetween the actual antigen and the primary antibody, the surrogateantigen targets can provide a valuation of the primary antibody'sefficacy. The primary antibody comes with an assay of the concentrationin mg/ml. However, the assay does not indicate what percentage of theassay represents complete IgG proteins. Since the density concentrationsof the surrogate antigen in the targets are known the capture of theapplied primary antibody concentration can be determined. Byextrapolation the antigen density of the co-resident tissue section canbe determined. The resolvability of antigen density on the tissue islimited to the cumulative displacement by the primary antibody(monoclonal or polyclonal), secondary antibody and enzyme gain of thesecondary stain. The concentration of the applied primary antibody isdependent upon the tissue type and always has some excess marginincluded. Thus, the assumption is that the primary antibody will bind toall the available antigen sites within its displacement limitations.

In some embodiments, the primary target density gradient array is aloading dot array of a universal surrogate antigen. In some embodiments,the loading dots in the array includes increasing or decreasing densityof the surrogate antigen. In some embodiments, the surrogate antigen isblended with one or more dummy proteins, such as dummy surrogateantibodies, and the increasing or decreasing concentration of thesurrogate antigen is achieved by altering the ratio between thesurrogate antigen and the dummy protein while maintaining the maximumtarget density.

In some embodiments, the control area 140 includes at least two arraysof surrogate antigen target. In some embodiments, the control area 140includes a first surrogate antigen target array including an anti-mouseantibody and a second surrogate antigen target array including ananti-rabbit antibody, both blended with a dummy surrogate antigen toform the desired reactive density while maintaining the maximum targetdensity.

Therefore, according to these embodiments, with the IHC or ICC steps thesurrogate antigen primary targets can evaluate the primary antibodyconcentration using the enzyme gain and antigen retrieval factorsdeveloped by the secondary targets.

Antigen Retrieval Targets

In some embodiments, the control area 140 includes an antigen retrievaltarget.

IHC and ICC staining assays often include an antigen retrieval (alsoreferred to as “AR”) process. AR process can be performed with a heatinduced epitope retrieval (HIER) method, or warm water antigen retrievalmethod. Depending on the method used as well as the implementation ofthe specific methods, significant amount of variation is introduced andthe results varies from practitioner-to-practitioner and slide-to-slide.Direct measurement of the AR buffer and the buffer temperature is oftennot accurate. However, without a proper control, practitioners oftenhave to assume blindly that the temperature and conditions of the ARbuffer are ideal. Therefore, the lack of control often results infailures of the AR process to slip into the final evaluation, resultingin misjudgments.

The most common issues in the AR process is under recovery or overrecovery of antigens. The under recovery condition occurs when the ARtemperature is too low, exposure time is insufficient, etc. The overrecovered condition occurs when the AR temperature is too high, theexposure to AR condition is too long, or the AR buffer condition is tooharsh (e.g., >pH 9.5 or <pH 5.5.)

Therefore, in some embodiments, the antigen retrieval target isconfigured to provide indication on under recovery, nominal recovery,and over recovery during the AR process.

In some embodiments, the control area 140 includes a 3D AR target(ARM3D) and a 2D target (ARM2D). 2D and 3D targets are described indetail in the section below.

In some embodiments, the ARM2D target includes a 50:50 mix of mouse andrabbit protein (or protein from other species) at 100% concentrationwith minimal formaldehyde fixation. In some embodiments, the protein isIgG. Because 2D targets generates less signal than corresponding 3Dtargets, and the ARM2D target has minimal fixation, staining of theARM2D target indicates an insufficient antigen retrieval.

In some embodiments, the ARM3D target includes a 50:50 mix of mouse andrabbit protein (or protein from other species) at 100% concentrationdeposited in a 3D scaffold that has been over fixed with formaldehyde.Because 3D targets generate more signal than corresponding 2D targets,and the ARM3D target is over fixed, the absence of staining of ARM3Dtarget indicates that the AR process was too aggressive.

In some embodiments, the antigen retrieval target further includes aprimary target gradient array or a secondary target gradient array inaddition to the ARM2D or the ARM3D targets. In some embodiments, theprimary target array or the secondary target array is the same as orsimilar to those as described above.

In some embodiments, the secondary target gradient array is a mouse orrabbit density gradient array. According to these embodiments, when 10%to no more than the 30% target dots of the mouse and rabbit gradientdensity arrays do not show visible staining, nominal recovered conditionis indicated. The degree of AR process can then be assessed by thenumber of low concentration secondary targets that are not stained.

2D/3D Targets

In some embodiments, the control targets, such as the primary target,the secondary target or the antigen retrieval targets include a 2Dtarget.

As used herein, the term “2D target” means that the peptide, protein,antigen, antibody or any other control target materials are deposited ona 2D plane on the slide.

2D targets are easier to deposit and are therefore relatively cheap tomanufacture. Therefore, 2D targets are used when cost is concerned.

In some embodiments, the control targets, such as the primary target,the second target or the antigen retrieval targets include a 3D target.

As used herein, the term “3D target” means that at least one of thepeptide, protein, antigen, antibody or any other control targetmaterials are applied to a support structure and the composite depositedin a 3D space on the slide.

In one or more embodiments, 3D targets are constructed by forming a 3Dscaffold on the slide, and depositing the control target materials onthe scaffold. In some embodiments, the 3D scaffold is a polysaccharidecluster. The polysaccharides form covalent bonds with the hydroxyl oramine groups on peptides or on the surface of the protein, which linksproteins together and anchors the target to the slide coating adhesive.In some embodiments, the control targets are fixed on the 3D scaffold byformaldehyde fixation. The target is then composed of both 2D and 3Dcomponents.

In one or more embodiments, the 3D targets are constructed fromsubmicron beads to which the primary or secondary target materials arecovalently bound. Formaldehyde fixation is then applied to stabilize theprimary or secondary target materials against normal antigen retrievalprocessing. The deposited target then consists of 2D and 3D components.

Comparing to 2D targets, 3D targets behaves more similar to the stainingsamples. Staining samples of IHC or ICC, such as tissue sections andcells, have heights. The height of the samples commonly ranges from 4microns to 10 microns. Antigen sites targeted in the staining can beanywhere on the exposed surface topology of the tissue section. As such,antigen sites in the sample can be planar at either top of bottomsurface of the section or anywhere along a sidewall of the tissue. Thus,the antigen on the sidewall of the sample is able to precipitatesignificantly more colorant from the chromogen than the 2D targets areable to. The inclusion of the 3D elements with the 2D elements creates astep offset that can be applied to the other 2D target dots to form avirtual 3D array. Experimentally, it was found that any 3D targetgreater than 0.5 micron would stain as darkly as the tissue section.

In addition, DAB, a reagent commonly used in the staining process, agesover a relatively short amount of time resulting the changes of colorand intensity. The instant inventors have found that in the DAB stained3D targets the color and intensity changes in a manner similar to thosein the sample.

Special Stains

In some embodiments, the control targets includes a special stain.

In some embodiments, the special stain includes Alcian Blue, AnalineBlue—Orange G Solution, Azan Stain, Bielschowsky silver stain, Brow &Benn—Gramm Stain, Cresyl Violet, DAB, Fontana Masson, Gordon and Sweet'ssilver staining, Grocett's Methanamine silver method, Hall's Bilirubinstain, Jones Methanamine silver method, Luxol Fast Blue, Luxol FastBlue—Cresyl Violet, Mucicarmine (Mayer's Method), Muller-Mowry colloidalIron, Orange G, Nuclear Fast Red, PAS with Diastase Digestion, PeriodicAcid Schiff (PAS), Phosphotungstic Acid, Haematoxylin, Picro Sirius Red,Toluidine Blue Acidified, Trichrome—Gomoris One-Step,Trichrome—Masson's, Victoria Blue, Von Kossa, Weigert's ResorcinFuchsin, Weigert's Iron Haematoxylin, Zell—Neelsen Method, orcombinations thereof.

Imaging Reference Target

In some embodiments, the control area 140 further includes an imaginingreference target. In some embodiments, the image target includes a blacktarget or targets, a white target or targets, or a clear target.

Digital imaging of microscope slides containing stained biomaterials isevolving to perform prescreening and potentially full diagnosticdetermination on the stained materials. In some embodiments, after thestaining process, the slide is subject to an imaging process duringwhich the light illumination level or exposure are adjusted so that thedigital image is not in compression at either the white or blackboundary. In another approach black and white targets are located wherethe label is expected to be positioned. The underlying assumption isthat the white and black targets represent the extremes that the levelof staining signals can reach. However, in doing so there is compressionin the digital scale as the black is much blacker and the white muchwhiter than can realized by the staining of a tissue section.

In some embodiments, the imaging reference target is printed on theslide. In some embodiments, the imaging reference target include a pairof the black target and the white target. In some embodiments, imagingreference targets are printed paint deposits which are non-reactive tothe reagents used in the staining process.

In some imaging systems, only transmitted light is used (illuminationfrom the bottom of the slide). In other imaging systems, bothtransmitted light and reflected light (illumination from the top side ofthe slide). A third clear reference target supports reflected lightillumination. Thus, for transmitted light illumination the black/whitereferences are the black pigment target and the clear targetrespectively, while for reflected light illumination the black/whitereferences are the clear target and white target respectfully. See FIG.12 for an example of the single antibody slide and FIG. 13 for a dualantibody slide. The black/white targets are actually a string of dots,could alternatively be a bar that serve an additional function asbumpers to protect the target dots from damage when the slide isdispensed in an inkjet printer. Inkjet printers dispense slides from thebottom of the magazine. The bumpers ensure that the remaining stack ofslides cannot scrape the paraffin coating or targets on the slide. Thesesame bumps also ensure that should the slides be shipped in a packedform that the paraffin layer will not adhere to the bottom of anadjacent slide.

Imaging with transmitted light results in a misrepresentation of theantigen density on the tissue. This occurs because the stained tissue isnot a monolithic slab where all antigen sites are on a single plane.Rather, the tissue can have antigen sites anywhere within its thickness.The secondary stain produces colorant particles that continue toprecipitate until the particles cover the enzyme site and blockcontinued precipitation. Should an antigen site be on the sidewall ofthe tissue section, the enzyme will never become covered and theprecipitation will over express the antigen presence. With transmittedlight the ‘darkness’ occurs because the precipitated colorant isthicker. In contrast, reflected light only ‘sees’ the top surface of thecolorant pile resulting in a more accurate representation of antigendensity.

In some embodiments, the white target has a color close to perfectwhite. Because a perfect white color is difficult to obtain, in someembodiments, the color of the white target is 5-10% away from perfectwhite. White colors less than 5% away from perfect white is difficult toproduce, and colors more than 10% away from perfect white are sometimesnot accurate enough as a control for staining signal strength.

In some embodiments, the white target includes a white pigment. In someembodiments, the white pigment is a metal oxide or a metal sulfatepigment that is stable with the passage of time when not left exposed tostrong light. In some embodiments, the white pigment includes aluminumoxide, titanium oxide, or barium sulfate. In some embodiments, the metaloxide or the metal sulfate are in the form of beads.

In some embodiments, the black target includes a black pigment. In someembodiments, the black pigment includes a carbon based pigment. In someembodiments, the black pigment includes a carbon dust. In someembodiments, a diameter of the carbon dust is less than 2 microns.

In some embodiments, the imaging reference target includes ananhydride-based epoxy. In some embodiments, the imaging referencetargets are produced by mixing pigments with the anhydride-based epoxy,and curing the anhydride based epoxy. Matching of the epoxy binder andthe pigment are not particularly limited, a long as there is a goodwetting between the pigment and the epoxy binder.

In some embodiments, the anhydride based epoxy includes an anhydridecatalyzer that is able to eliminate unreacted amines of an amino-silanebased catalyzer. Free amine groups capture both biomaterials and some ofthe staining reagents. As such free amine causes non-specific stainingin the IHC and ICC process. Thus, the elimination of unreacted aminesreduces non-specific staining.

In some embodiments, the anhydride-based epoxy paint includes ananhydride catalyzer, such as methyl tetrahydrophthalic anhydride ordiphenyliodonium hexafluroro arsenate.

In some embodiments, curing the anhydride-based epoxy includingsubjecting the anhydride-based epoxy to a UV light, such as a UV lightincluding a wavelength of 365 nm. In some embodiments, curing theanhydride-based epoxy paint further includes subjecting the anhydridebased epoxy paint to heat, thereby allowing the epoxy to cross-link. TheUV initiated anhydride-based epoxy and companion reagents thereof arenot particularly limited. One of ordinary skill in the art is able toidentify desirable anhydride-based epoxy by performing a search ofproducts by anhydride producing companies.

In some embodiments, the imaging reference targets including theanhydride-based epoxy is deposited on the slide prior to the depositionof the primary targets, the second targets, or the antigen retrievaltargets. The reason is that the heat treatment in the curing process ofthe anhydride-based epoxy can damage biomaterials included in theprimary targets, the second targets, or the antigen retrieval targets.In some embodiments, the anhydride-based epoxy is cured by UV, and thedeposition of the imaging reference targets can happen after depositionof the primary targets, the second targets, or the antigen retrievaltargets as UV is less likely to damage peptides and proteins.

In some embodiments, the imaging reference target formulation is free ofa surfactant, thereby preventing the ink/paint to be reactive to therange of stains and reagents these slides can experience.

In some embodiments, the imaging reference target is printed on theslide by a pad stamp. In other embodiments, the imaging reference targetis printed by a syringe, because the syringe is able to control a sizeof target deposition.

Using the imaging reference targets, digital analysis is optimizedbecause the all of the digitization range is within the slidesdefinition of black/white versus the use of perfect black and whitereferences. The digital analysis assists with interpolation of a colorof the control targets in order to more precisely determine an amount ofantigen present in the stained sample. The image reference target isused in order to assist with calibration of the digital analysis inorder to provide a reference point for measuring an amount of reactionat the control targets.

Method of Depositing Control Targets

In some embodiments, the instant specification is directed to a methodof depositing control targets on a process recording slide. In someembodiments, the slide and the control targets are the same as orsimilar to those as described above.

In some embodiments, the method of depositing control targets includingdepositing a primary control target or a secondary control target on theslide.

In some embodiments, depositing the primary control target or thesecondary control target includes: preparing a solution of the controltarget at a predetermined concentration; fixing the control target; andprinting the solution onto the slide.

In some embodiments, preparing the solution of the control targetincludes preparing a solution including the control target and apolysaccharide to function as the linker between the proteins and theslide adhesive.

In some embodiments, fixing the control target includes fixing thecontrol target with formaldehyde. The formaldehyde crosslinks thecontrol targets so that the control targets can withstand normal antigenretrieval protocols.

In some embodiments, the control targets are crosslinked. Within tissue,proteins are bound to other parts of the tissue that tend to keep theproteins from diffusion and denaturation. Since the control targets inthe solution includes loose proteins, the control targets are moresensitive to heat and pH and, as such, tend to diffuse and denature onthe slide, especially when going through the AR process in IHC or OCC.

In some embodiments, the primary targets are prepared as follows:

-   -   Form a set of the primary protein master dilutions of 1 ml with        a concentration of 45 ug/ml. Dilute the Donkey-anti-Mouse,        Donkey-anti-Rabbit, and Donkey IgG (H&L) proteins with dH2O as        needed to realize the 45 ug/ml concentration. Typically, these        proteins are between 1 and 10 mg/ml in concentration as        purchased. Add in 10 ul of Thimersol as a fungal growth        inhibitor.    -   Fix each master dilution with 10 ul 0.2% formaldehyde for 1-4        hrs at 40-60° C.    -   Add in 30 ul of 0.45% concentration amylose as a linear        polymer(has Thimersol added as a fungal growth inhibitor) and        mix for 30-min    -   Add in 20 ul of 0.1M ammonium bicarbonate to quench any        unreacted formaldehyde    -   Use the master protein solutions to form the target mixtures of        Mouse with Donkey and Rabbit with Donkey, wherein each target        contains 700 ul of the master protein solutions.

In some embodiments, the secondary targets are prepared as follows:

-   -   Form a set of the secondary protein master dilutions of 1 ml        with a concentration of 45 ug/ml. Dilute the Mouse, Rabbit, and        Donkey IgG (H&L) proteins with dH2O as needed to realize the 45        ug/ml concentration. Typically, these proteins are between 10        and 60 mg/ml in concentration as purchased. Add in 10 ul of        Thimersol as a fungal growth inhibitor.    -   Fix each master dilution with 10 ul 0.2% formaldehyde for 1-4        hrs at 40-50° C.    -   Add in 30 ul of 0.45% concentration amylose as a linear polymer        (has Thimersol added as a fungal growth inhibitor) and mix for        30-min    -   Add in 20 ul of 0.1M ammonium bicarbonate to quench any        unreacted formaldehyde

Use the master protein solutions to form the target mixtures of Mousewith Donkey and Rabbit with Donkey, wherein each target contains 700 ulof the master protein solutions.

In some embodiments, the prepared primary or secondary targets, or imagereference targets are printed onto the slide in one print cycleaccording to the follows:

-   -   Print the target solutions onto an adhesive coated microscope    -   Air dry at 60° C. until all water has been evaporated then cool    -   Apply the paraffin-solvent mixture by spray over the printed        target arrays    -   Reflow the paraffin to complete sealing of the targets arrays        and drive off the solvent. Thus, returning the paraffin into its        hardened and solid state.

Method of IHC or ICC Staining

In some embodiments, the instant specification is directed to a methodfor immunohistochemical (IHC) or immunocytochemistry (ICC) staining. Insome embodiments, the instant specification is directed to a method forimmunohistochemical (IHC) or immunocytochemistry (ICC) staining usingthe process record slide as described above.

In some embodiments, the IHC or ICC staining process includes: embeddinga fixed sample into paraffin, and removing the paraffin to expose theantigen sites within the cellular structure of sample.

In some embodiments, removing the paraffin comprises: warming theparaffin into a semi-liquid state at a temperature ranging from 65 to 75degrees C. for 3-10 minutes, then liquefying the semi-liquid paraffinwith an aliphatic solvent, such as xylene or xylol followed by arehydration sequence of anhydrous ethanol, 95% ethanol, 70% ethanol, 50%ethanol, and a salt-based buffer solution.

In some embodiments, the IHC or ICC staining process further includesremoving the formaldehyde fixing to expose the antigen sites in thesample. In some embodiments, removing the formaldehyde fixing includingperforming a heat induced epitope retrieval (HIER) process, orperforming a many cycle warm water antigen retrieval process.

In some embodiments, the HIER process includes breaking the Schiff basebond between the formaldehyde and tissue by subjecting the sample toheat in the presence of water. In some embodiments, subjecting thesample to heat comprises subject the sample to a temperature rangingfrom 89° C. to 95° C. In some embodiments, the sample is exposed to abuffer reagent when being subjected to heat. In some embodiments, thebuffer reagent has pH ranging from 6 to 10, such as from 6 to 9. Thechoice of the pH of the reagent depends on the type of the sample, suchas tissue type.

In some embodiments, the water-based antigen retrieval process includessubjecting the sample to a temperature about 10° C. higher than themelting temperature of the embedding paraffin, which ranges from about60° C. to about 65° C. In some embodiments, the water-based antigenretrieval process includes subjecting the sample to soap and successivewashes to dissolve and remove the paraffin.

It should be noted that operator errors or processing defects in theparaffin removal and fixation recovery can block the subsequent stainingprocess and result in a false negative result. Although such falsenegative result can be detected by reviewing the staining results of thecontrol targets in the process recording slide, the operator errors orprocessing defects would nonetheless result in waste of samples, timeand resources, and should be avoided.

At this point, the antigen sites are exposed and the stain reagents canbe applied to produce a signal indicating the presence and amount of thetargeted antigen.

In some embodiments, the IHC or ICC staining process further includesapplying one or more primary antibodies. If more than one primaryantibody is applied, the antibodies need to be from different animalspecies to avoid cross-reaction by the secondary antibody. For example,if two primary antibodies are used and the first one is a mouseantibody, then the second primary antibody needs to be a non-mouseantibody like a rabbit antibody. According to these embodiments, the oneor more primary antibody will bind to the matching antigen sites in thesample, as well as to the primary control target such as theprotein/peptide antigen or the surrogate antigen. In some embodiments,the primary antibody includes an antibody targeting ER, PR, Her2, orKi67.

Refer to FIG. 11, in some embodiments, the primary antibody isconjugated with a reporter, such as a chromogenic reporter, such as anenzyme chromogenic reporter. According to these embodiments, nosecondary antibody is needed.

In some embodiments, the primary antibody is not conjugated with areporter. According to these embodiments, a secondary antibody targetingthe primary antibody is applied. In some embodiments, the secondaryantibody is conjugated with the reporter.

Refers to FIG. 11, in some embodiments, to further amplify the signal ofthe antigen, a multistep signal amplification process is carried out.Examples of multistep signal amplification process includes: 2× gain forenzyme-labeled tertiary antibody reacts with enzyme-labeled secondaryantibody, 3-step indirect; 2× gain for APAAP immune complex reacts withsecondary antibody. In some embodiments, the primary antibody and theantibodies of the immune complex are made in the same species. Thesignal amplification process includes: LAB or LSAB, Enzyme-labeled(strept) avidin reacts with the biotinylated secondary antibody; >10×gain for CSA technology, Streptavidin-enzyme complex on biotintylatedsecondary antibody on primary antibody; >10× gain for a polymercontaining 10 secondary antibodies and 70 enzyme sites bound to a singleprimary antibody.

In some embodiments, the reporter is an enzyme capable of causing achromogen precipitation when a substrate is present. All arrive at thesame end state of a chromogen precipitation.

In some embodiments, the reporter enzyme includes horseradish peroxidase(HRP), alkaline phosphatase (AP) or glucose oxidase.

In some embodiments, one or two of, three commonly used secondary staingroups and one of several counterstains are used: Horseradish peroxidase(HRP), alkaline phosphatase (AP), glucose oxidase and nuclearcounterstains.

In some embodiments, the chromogen includes 3,3′-Diaminobenzidine (DAB),Amino-9-ethyl carbazole (AEC), DAB+Nickel enhancer, Fast Red, TMB,StayYellow, BCIP/NBT, BCIP/TNBT, Naphitol AS-MX phosphate+Fast Blue BB,Naphihol AS-MX phosphate+Fast Red TR, Naphitol AS-MX phosphate+newfuchsin, StayGreen, NBT, or combinations thereof.

In some embodiments, the substrate and reporter enzyme combination, aswell as the result color is the follows:

HRP (Horseraddish peroxidase) DAB (3,3′-Diaminobenzidine) >> Brown toRed Brown AEC (3-Amino-9-ethylcarbazole) >> Red DAB + Nickel enhancer >>Black TNB (3,3′,5,5′-Tetramethylbenzidin) >> Blue Stay Yellow >> YellowAP (Alkaline Phosphatase) BCIP/NBT >> Blue(5-bromo-4-chloro-3-indolyl-phosphate)/ (nitro blue tetrazolium)Naphthol AS-MX phosphate + Fast Blue >> Blue Naphthol AS-MX phosphate +Fast Red >> Red Naphthol AS-MX phosphate + new fuchsin >> RedStayGreen >> Green GO (Glucose oxidase) Nitro blue tetrazolium chloride(NBT) >> Blue to purple

In some embodiments, the IHC or ICC staining process further includes acounterstaining, such as a nuclear counterstaining. In some embodiments,the nuclear counterstaining uses one or more nuclear stains, such ashematoxylin that generates a blue color.

The choice of the staining substrate or compound can be made based onthe color requirement, stability requirement, and regional regulatorystandards.

For example, DAB is commonly used in countries such as the USA and Chinawhile AEC is commonly used in other countries. DAB is often used overAEC due to the fact that the brown-red color generated by DAB has highersaturation as compared to the red color of the AEC. However, AECstaining is more stable than DAB staining. Experiments show that theoriginal DAB ages significantly over a short period of time, such thatthe color saturation drops noticeably within a 4-hour span. Newerversions of DAB incorporate stabilizers that extend the stability of theDAB from hours to days. DAB also has the propensity to be washed outduring subsequent buffer wash cycles. AEC, on the other hand, remainsstable for weeks to months.

Regulatory standards throughout the world seek, or insist, thatvalidated controls be used to check reagents, methods, andinstrumentation for processing of tissue sections and loose cells oncesuch a technology becomes viable and available. Such regulatory controlshave long been in place for hematology and clinical chemistry tovalidate the results and for quality assurance. The result of thecontrols testing is plotted in the form of a Levey-Jennings chart(Westgard et al. 1981). Westgard J, Barry P, Hunt M, Groth T (1981) “Amulti-rule Shewhart chart for quality control in clinical chemistry”.Clin Chem27:493-501.

Antigen Imaging Scale Extrapolation

In some embodiments, the method of IHC or ICC staining further includesestimating an antigen concentration in the co-resident patient sample byextrapolating the stained result of the primary and secondary targets.

In some embodiments, the method of IHC or ICC staining further includesdetermining the Process QC of the IHC or ICC staining steps byextrapolating the stained result of the primary and secondary targets.

Because the amount of the control targets deposited on the slide ispredetermined, knowing the molecular mass, the number of control targetmolecules of each protein type in the deposit, the target's area, andthe slide coating's porosity an active surface protein density of thetarget can be calculated.

The applied concentration, dispensed volume, and surface area on slideexposed to the reagent of primary antibody are known. It can bereasonably assumed that during the exposure time of the reagent thatmost of the suspended antibodies will have fallen down and been capturedby receptive antigen sites. Only those that fall directly over antigensites will become captured and the balance will be washed away by abuffer wash step. Thus, the deposited antibody concentration can beestablished under proper conditions, for example, when the concentrationis greater than 25% above cutoff and less than 25% from saturation. Asused herein, the term “cutoff” is defined as insufficient target sitedensity to capture the applied the protein concentration; saturation isdefined as a concentration at which not all of the applied protein couldbe captured.

Knowing the primary dilution ratio, the correct primary target densitytarget can be chosen and the primary concentration can be validated.

In one embodiment of the present invention, each secondary and primarytarget is a mix blend of [(mouse or rabbit IgG)+(donkeyIgG+crosslinker+fungal inhibitor)] or [(KLH with antigen A or KLH withantigen B)+(unconjugated KLH+crosslinker+fungal inhibitor)] or surrogateantigen [(Donkey-anti-Mouse IgG or Donkey-anti-RabbitIgG)+crosslinker+donkey IgG+fungal inhibitor]. Each dot has the samevolume of total proteins, but the mix ratio is adjusted slightly so thatthe atomic masses are different between the proteins composing aspecific target;

The molecular weight of some exemplary proteins are listed below:

Mouse IgG=155 kDa

Rabbit IgG=150 kDa

Donkey IgG, Donkey-anti-Mouse, and Donkey-anti-Rabbit=160 kDa

Protein A=42 kDa

Protein G=58 or 65 kDa

Protein A/G=50 kDa

Protein L=76 kDa

Chicken IgY=180 kDa

KLH subunits: KLH1 and KLH2=350 and 390 kDa, respectively.

In another embodiment of the present invention, the 2D secondary targetgradient includes stepped dilution increments of 1 to 1000:1. In someembodiments, the dilution follows a −20 log (dilution) profile, whereinthe dilution increments in −3 dBd steps. The terms −20 log(dilution)=dBd, both describe the dilutions on a semi-logarithmic basisin order to linearize the data so that modifying terms can be easilyapplied. The term (dillution) refers to the dilution X where X is [1 . .. 1000] equating to 1:1 to 1,000:1. The term dBd is defined as decibelsof dilution or dilution strength. The modifying terms include antigenretrieval damage, enzyme gain, or primary antibody reagent dilution;

The secondary stain incorporates an enzyme gain function between 1 and20×, which is a function of the construction of the stain reagent.Therefore, as the gain rises the lower concentration secondary targetwill shift into saturation whereas when the gain drops to one only thehigh concentration secondary targets will be visibly stained;

Because of the considerable size difference between the secondary andconjugated primary target proteins based on KLH subunits with peptides,the dummy diluent must be un-activated KLH units as the peptides do makefor a noticeable change in mass to the KLH subunit.

The expected binding capacity of the KLH subunit to Protein A, G, andA/G is predicted to be between four and six proteins. For a suitabledummy the KLH needs to be joined with Chicken IgY, which is completelynon-reactive to any of the Protein A, G, A/G, or L proteins. While thechicken IgY is nearly 4× bigger than Protein A/G then will be less boundto the KLH so the end result is about the same. Either choice puts themass to 650 to 700 kDa. As detailed below for mouse and rabbit IgG theKLH subunit mass figure can be plugged in and the calculationsperformed.

In another embodiment of the present invention, the primary targets canbe fabricated from Protein A, G, or A/G. The primary antibody isdominantly based on either a mouse IgG or rabbit IgG host Fc domains.However, the FcyRI sites are found on mouse IgG1 and rabbit IgG. ProteinA binds strongly to rabbit IgG, but weakly to mouse IgG1. Protein Gbinds strongly to rabbit IgG and medium to mouse IgG1. Protein A/G bindsstrongly to rabbit IgG and medium to mouse IgG1. Protein L bindsstrongly to mouse IgG1 and weakly to rabbit IgG in the Fab light chainversus the FcyRI of the others. In some embodiments, Protein G or A/Gare used. Both appear as dipole structures, wherein they support only asingle connection to a primary antibody.

Protein A, G, A/G and L bind to most placenta mammals IgG, which meansthat the blocking step before the application of the secondary stain kitwill not work if BSA (bovine serum albumin) is used as BSA will not bindto these proteins. In particular, goat is blocked from binding toProteins A, G, A/G or L. This is different from other approaches, asmost secondary stain kits use goat as a host protein at some step. Thus,it is strongly recommended that a blocker be based on the equine family:horse, donkey, taper, and rhino IgG proteins. The equine family splitaway genetically early from the placenta mammals, enough so that it islargely non-reactive to the secondary stain reagents, but will bind toProtein A, G, and A/G. sufficiently to block the unreacted primarytarget sites.

Because the binding connection to the primary antibody always leaves oneof the two FcyRI sites open for reacting with the secondary antibody,the captured primary antibody must be functionally intact with at leastone FcyRI site available to support the secondary staining. Thus, thecaptured primary antibody that becomes stained represents the actualapplied concentration versus the primary's assay/dilution figure.

With an average primary antibody atomic mass is 150 kDa, the molecularweight of a single antibody molecule is 150 kDa (1.6605×10¹²), whichequals a weight of 249×10⁻¹²ng. Assuming a single area of the slide isthe only part exposed, then the amount of applied primary reagent can bedetermined. For example, when a closed capillary gap having an insidedimensions of 20.3 mmsq×0.14 mm is used, the volume is 57.2 μL. Ratiofor a target area of 1 micron, which yields 2.832 nl of the appliedprimary antibody reagent;

The primary antibody reagent is diluted from a concentrate to anintermediate dilution of 10 ug/ml. The intermediate dilution is thendiluted, from 1:1 to 1000:1, for application onto the slide. Thisresults in a deposition of 31.5 to 7.08 antibodies onto a 1 micron areafor a dilution of 1:1 to 25.1:1 respectively.

To ensure 100% capture ability the primary target should have a safetyfactor ranging from 100 to 1000×. When the 1000× option is chosen theprimary target needs to contain 4×10⁶ antigen sites. While the KHLsubunits are bigger than the applied antibodies, the increase is notenough to change the number of captured antibodies beyond 1:1. Each KLHsubunit has an average atomic mass of 370 kDa which equates to a weightof 614.4×10⁻¹²ng.

The volume of a protein molecule can be approximated from the molecularweight of the protein and an average protein partial specific volume(partial specific volume=volume/molecular weight). The average ofexperimentally determined partial specific volumes for soluble, globularproteins is ˜0.73 cm³/g. This value varies from protein to protein, butthe range is narrow. The equation reduces down to a protein volume of˜(1.212×10³×MW) nm³. Thus, for the KLH subunit the individual volume is448.44 nm³. Assuming that the protein is a sphere, the diameter of thesphere is calculated as 0.132×MW^(1/3) in nm. According to thiscalculation method, the diameter of a KLH subunit is about 9.436 nm;

For a control target dot having a diameter of 1 mm and includes amonolayer of the KLH subunits, 11.237×10²⁷ proteins are needed. For theactive target density of 4×10⁶ proteins the minimum dilution ratio is1:2.8×10²¹. In practical terms, any dilution approaching 1:1000 isworkable as the evaluation of the primary antibody is dominated by itsactive protein concentration. Thus, the target density is only limitedby its low concentration floor value.

In one embodiment, the secondary target arrays are stepped dilutionincrements of 1 to 1000:1. A linear slope for the dilution occurs asdBd=−20 log (dilution). For the dilution range of 1 to 1,000:1, thesemi-log range is 0 dB to −60 dBd. In some embodiments, −3 dB steppingdilution of the secondary target results in a dilution series of −0, −3,−6, −9, −12, −15, −18, −21 dBd.

The secondary and primary target arrays are both semi-reversibly fixedand therefore undergo a smaller degree of degradation comparing to thesample or the AR targets during the AR process. The degradation comesfrom protein segments that break free rather than complete proteins. Insome embodiments, as the AR process continues to act on the proteintargets and the sample section the AR damage considered as the gradientscale pattern shifts towards the 100% position. On the other hand, thesecondary enzyme gain causes the gradient array to shift towards the 10%position. In some embodiments, the enzyme gains are 1, 2, 4, 5, 8, 10,15, or 20. This translates into shifting the secondary array towards the10% target by:

1. 20x  all targets shift −26 dBd 2. 15x  all targets shift −23.52 3.10x  all targets shift −20 4. 5x all targets shift −13.98 5. 4x alltargets shift −12.04 6. 2x all targets shift −6.02 7. 1x only 2D 100%dot near black

Typically, AR damage that shifts the secondary array towards the 100%position by three or more dots is considered to be excessive and thestaining should be redone using a higher enzyme gain secondary stain kitor a higher concentration of antibody.

The primary antigen target color density is thus the collective sum ofthe antibody concentration times the enzyme gain of the secondary stainkit. While the secondary target density is only that of the enzyme gaintimes the secondary target protein concentration;

Depending on the digital imaging system, changes in the illuminationintensity will shift the dynamic range of the image into saturation(getting darker) or cutoff (getting lighter). These changes shift theantigen color scale while the antigen density numeric scale will not.Thus, the numeric scale is independent and the color scale dependent onthe illumination intensity.

In one embodiment of the present invention, see FIG. 13, within thecontrol area 140, the aforementioned secondary protein target arrays areformed as two lines: one of mouse IgG and the other rabbit IgG mixedwith a dummy IgG blood serum protein to form a five or more membergradient density series that progresses from a maximum density to aminimum density in a −20 log (dilution) linear slope, wherein thedilutions may range between 1:1 to 1,000:1 after the initial 1000:1dilution.

In another embodiment of the present invention, the aforementionedsecondary target arrays are formed as three concentrations of 33, 16.5,and 4% of the mouse IgG or the rabbit IgG mixed with donkey IgG.

In another embodiment, in the last process step, those antigen sitesidentified become colored by chromogen precipitation. Thus, the mouseand rabbit target array reflects the −20 log (dilution) linear slope ofsecondary stain kit chromogen precipitation or the target arrays reflectthe secondary stain reactivity and enzyme gain.

In another embodiment, the solution for the method for forming theprimary target density gradient array is predicated on successfullycomposing the target mixtures, depositing them onto the adhesive coatedslide, and having a covalent bond between the adhesive and the targetmaterials.

In another embodiment, deducing that the target arrays are successfullydeposited and the both the primary and secondary stain reagents performreasonably, a curve fitting between the data sets can be done bycomputer algorithm. In another embodiment, the primary stain is selectedfrom any IHC or ICC approved antibody that including a mouse or rabbithost protein that is not also conjugated to a fluorescent marker orintegrated with an enzyme site (such as HRP or AP). In anotherembodiment, the secondary stain is include secondary stains havingenzyme gains of 1× through 25×, that are each uniquely independentbetween mouse and rabbit, which each use a different color chromogen.

In another embodiment of the present invention, it is pertinent to notethat the performance result in an absolute basis on one slide may not beidentical to another slide done at another time. This comes from thefact that the secondary stain kits vary in performance lot to lot asdoes the primary conjugated primary antibody. However, the results ofthe process record slides can be validated by the control targets andprovide equivalence to another slide done using different stainreagents.

In some embodiments, the primary antigen concentration scale is thenapplied to the co-resident tissue section to access the tissue sectionfor detecting cellular defects, such as cancer.

Various embodiments are described herein as examples. It will beapparent to those skilled in the art that various modifications may bemade and other embodiments can be used without departing from thebroader scope of the invention(s) presented herein. These and othervariations upon the exemplary embodiments are intended to be covered bythe present invention(s).

EXAMPLES

The following examples are presented in a way to the illustration of theinvention and should not be construed to limit the scope of theinvention in any manner.

Example 1 Paraffin Shield Coating with Spray Application Method

The surface of the slide is sprayed over with low airflow. In someembodiments, a low liquid to air mix is used. The mixture is sprayedonto the slide, through a mask, to cover the control targets. Typically,1-2 passes are performed to form a layer having a thickness of less than5 microns without the need to reheat to flow the paraffin seal. Theparaffin mixture reservoir and spray head are both heated to slightlyhigher than 56° C. to ensure the paraffin is sprayed as a fluid andremains as a fluid while in flight to the slide. Spray coverage from thehead is nominally 0.375″ in width. The deposited paraffin mixture isreflowed by raising the ambient heat to 60° C. for one minute, whichdrives out the solvent and enables the paraffin to return to a hardenedstate when cooled.

Example 2 Paraffin Shield Coating with Screen Printing Method

A stainless steel printing screen is heated by passing an electricalcurrent through the wires of the screen from two parallel sides. Thetemperature of the screen is slightly below the melting temperature ofparaffin so that paraffin paste does not weep through to the bottom sideof the screen. At such temperatures, the paraffin behaves closer to apaste than a liquid. The deposited paraffin is reflowed by raising theambient heat to 60° C. for one minute. Any solvent in the appliedparaffin mixture is then driven out and the paraffin returns to hardenedstate.

Example 3 Paraffin Shield Coating with Ink jet Method

An inkjet head incorporating an integrated heater is to be used to keepthe paraffin in the liquid state. An aliphatic solvent, Zylol forexample, is used to change the paraffin from a solid to a liquid. Thedeposited paraffin mixture is reflowed by raising the ambient heat to60° C. for one minute. The solvent in the applied paraffin mixture isthen driven out and the paraffin returns to hardened state.

Example 4 Paraffin Shield Coating with Roller Transfer Printing Method

A heated roller pulls up a film of paraffin from a heated reservoir. Theroller then transfers the film of paraffin onto the slide much in thesame fashion as a painting a wall with a napped roller. The depositedparaffin is reflowed by raising the ambient heat to 60° C. for oneminute. The solvent in the applied paraffin mixture is then driven outand the paraffin returns to hardened state.

Example 5 Antigen Retrieval Exposure Time Versus Degradation to a SimpleLogarithm Series of PRS Secondary Targets

The test study sought to verify that the changes in AR exposure timewould be seen in 2D secondary targets and the AR targets.

The expected result was a linear slope of antigen retrieval exposuretime and signal strength reduction. Indeed, when the time taken for theAR buffer to reach above 89° C. is taken out of consideration, theplotted slope was linear. Using an 8-bit digitization with the PRSblack/white targets to set the white balance and contrast optimally, aslope of 1.3 lsb/minute, +/−0.2 lsb was obtained. The experiment showsthat the signals of the AR targets and the secondary targets reduces ina linear manner upon AR exposure for 20 minutes past the 89° C. timemark. After the 20 minutes mark, due to that 50% or more of the controltarget was under serious stress, the usefulness of the secondary targetswas compromised.

Example 6 Consistency of the Secondary Targets

The test study had two factors being explored:

I. Dot-to-dot comparison among a batch of slides assembled using asingle lot code secondary protein source and dilutions.

II. Dot-to-dot comparison among slides assembled using different lotcode secondary protein and dilutions.

Tests were performed using 100% and 40% target formulations. One hundredslides were printed and all processed with an avidin-biotin complex(ABC) type mouse and rabbit secondary stain kit from Scytek. Antigenretrieval was not performed as it adds an additional variable. Thedistribution of both was within 1.5%.

Example 7 Selection of Dummy Protein

Ten different secondary arrays were constructed using two different lotsof mouse, rabbit, and bovine IgG proteins. The arrays were constructedwith a dilution of 100, 40, and 20% of the IgG proteins. Thedistribution was within 1.5% for the 100 and 40% dilution groups. The20% dilution groups showed an unexpected increase in stain strength. Theinstant inventors discovered that the issue was due to an unexpectedinteraction between the bovine IgG and biotinylated goat-anti-polyvalentreagent from the ABC stain kit. The problem was solved by replacing thebovine IgG with donkey proteins. The test was repeated and the 20% groupis now within 1.5%.

Example 8 Quality Control Using Secondary Logarithm Series PRS

In QC mode, the co-resident targets provide IHC process feedback as isillustrated in FIG. 6.

Referring to FIG. 6, there four arrays of (50:50 mix of mouse & rabbit)secondary targets exposed to the degree of antigen retrieval performedfrom within nominal, mildly over, over, and excessively over (5, 10, 30,and 40% respectively). The staining results come from four differentslides representing each of the listed conditions above. The antigenretrieval process seeks to unmask the antigen sites by reversing theSchiff base bond between the formaldehyde and proteins. The speed atwhich the antigens become exposed depends upon the temperature of thereaction. As the temperature is increased, the opportunity occurs fornucleated boiling. The nucleated boiling causes physical damage to boththe tissue and protein deposits. Ideally, the antigen retrieval (AR)activity is uniform through the slide, however, in practice this isoften not the case and areas having slightly more or less antigenretrieval activity exists. If such un-uniformity of antigen retrievalactivity is ignored, the following can be used to indicate that theslide will be usable for diagnostic determination.

If the AR is insufficient or excessive, the secondary array may not beable to reflect the failure. The two AR targets however, will signal theinsufficient or excessive failure conditions.

-   -   a. Low AR is seen as the 2D/3D under fixed and 2D over fixed        targets are both black. The secondary arrays will appear as        perfect with no AR shifting left of the targets. Low AR activity        can occur from the following situations in the IHC stainer:        -   i. AR Heater not working or set well below 80° C.        -   ii. AR buffer has a neutral pH 7, rather than 6 or 9        -   iii. Exposure time too short    -   b. High AR is seen as the 2D/3D under fixed is very bleached and        the 2D over fixed target is less than 50% black. The secondary        arrays will be largely bleached out as well. High AR activity        can occur from the following situations in the IHC stainer:        -   i. Heater operating at temperature >95° C.        -   ii. Exposure time too long    -   c. Chromogen precipitation error can arise under two situations:        -   i. If at the high concentration secondary targets the stain            intensity dips rather than is at maximum darkness. The            secondary array should be always increasing vs. site            density. If not then the chromogen precipitation has            exhausted the secondary reagent kit capacity. The solution            is to increase the primary antibody dilution (same as            reducing the antibody concentration).        -   ii. The chromogen reagent has deteriorated since being            activated (often occurs with DAB). The solution is to use a            new DAB mixture.

The staining can experience saturation or cutoff as a function of theconcentration of the primary antibody and the enzyme gain of thesecondary stain kit. Saturation is when the density of the enzyme sitesexceeds the capacity to precipitate colorant from the chromogen. Inother words, the stain color is as dark as can be realized. Cutoffoccurs when the concentration of the primary antibody and enzyme gain ofthe secondary stain kit are too low, resulting in insufficient colorantprecipitation to be seen. The two factors cause the darkness of thesecondary line to shift to saturation (100%) or cutoff (0%). Based onFIG. 6, this movement is seen as the number of targets that are visible.As the secondary enzyme gain increases the 100% dot density shiftstowards the 0% position. The common enzyme gains are: 1, 2, 4, 5, 8, 10,15, and 20. These translate into shifting the secondary array towardsthe 0% position by:

20x  all targets shift −26 dBd 15x  all targets shift −23.52 10x  alltargets shift −20 5x all targets shift −13.98 4x all targets shift−12.04 2x all targets shift −6.02 1x only 2D 100% dot near black

If the primary target array is present an increase in secondary enzymegain shifts the stain density towards the low primary concentration dot.The same is true if the primary antibody concentration is increased. Theantigen retrieval process will cause both primary and secondary targetsto be degraded to some level, which reverses the shift towards cutoff.If at the end of the IHC staining there are three or more dots that havedisappeared the slide would be considered to have had excessive antigenretrieval duration, temperature, or both and too much antigen presencehas been lost on the tissue making diagnostic interpretation marginal.This decision is independent of the efficacy of the primary antibody asthe secondary staining is already been shown to be compromised. Nothingon the antibody step can overcome this damage level.

Example 9 PRS Tracks Illumination Level with Antigen Density Scale

Viewing a microscope slide through a conventional microscope issubjective in regards to the transmitted light illumination level. Inwhole slide imaging (WSI), the scanner uses a perfect white and blackhole to establish the white balance and contrast. Such is not the casewith manual microscopes. FIG. 10 illustrates the effect to the image asthe illumination level is too dark (−5% from optimal), optimal (+0), andtoo bright as in (+10 or +15%). When the light level is below optimalthere is compression of stain density. In terms of cancer stages thiscould shift the diagnosis one stage higher than it should be. When thelight level is above optimal there is bleaching of the image. In termsof cancer stages this could shift the diagnosis one stage lower than itshould be. The antigen color density and numeric ruler is developed fromthe primary and secondary targets and can be superimposed upon the WSIimage. The numerical scale is the independent term while the colordensity is the dependent term. When the antigen density color andnumeric ruler is applied to the WSI the numeric scale remains fixed asthe user shifts the illumination level up or down. The color densityscale on the other hand shifts as the illumination level changes. Theadvantage is that the user has the choice to shift the apparentillumination up/down to best ‘see’ features on the tissue image whilenever losing the numeric relationship to color density. This will alsobe functional as the magnification is changed.

Example 10 Construction of Antigen Density Ruler

According to some embodiments, two types of antigen density ruler aredeveloped.

1. Type A uses a secondary target array, which is based on theassumption that the primary antibody is always applied with less than10% excess antibody vs. tissue antigen sites.

2. Type B uses a primary target array (a primary antigen gradientdensity array).

Type A: Secondary Only Based Antigen Ruler

This form uses only the secondary target array. The passed ininformation that is imbedded in the 2D bar code includes the (a) primaryantibody data: host species for the antibody and dilution in -dBd (dBdis dB dilution) and (b) secondary enzyme gain.

The secondary gradient density target array is composed of knownconcentrations of secondary target proteins following an −3 dBddecrement between targets. The maximum concentration is chosen by theleast dilution that is used for the primary antibody. Users often takethe concentration specification provided by the antibody reagentmanufacturer and dilute to a constant intermediate concentration of 1ug/ml. From that all other dilutions are made as needed to accommodatethe different tissue types. In general, the second set of primaryantibody dilutions range between 1:1 and 1,000:1.

To accommodate the range of secondary enzyme gain the secondary arrayincludes of a wider range of dilutions. Thus, with −3 dBd steps thelowest dilution of the secondary array starts at 1,000:1 or −60 dBd,which is represented by SdBd (secondary dB dilution). The maximum of the8-dot series then becomes −0 dBd or 1:1. The action of the antigenretrieval degrades the secondary targets which is represented by ARdBd(antigen retrieval dB dilution). Each dot, one of eight, in thesecondary array represents an −3 dBd increment. The antigen retrievalloss for the loss of two targets (no longer visible) would be +6 dBd.This means the secondary array is (−S+AR)dBd for the 2D targets or [+6to −54 dBd]. The antibody concentration and the secondary enzyme gain isnow factored in. The antibody concentration would be AdBd, while theenzyme gain is EdBd. Thus, the secondary array would be (−S+AR−E)dBd,while the tissue would be (+AR−E+A)dBd. The next factor that applied isthe 100% 2D to 3D differential. The stain difference between the 3Dobjects in the 100% 2D/3D target and the 100% 2D represents thesecondary stain chromogen precipitation constant, which is used toassign the color density to the numerical scale and is assigned to DdBd.The difference in color density is applied to each of the 2D targets inthe array. Thus, the 2D array presents in stain color density as(+AR−E+A+D)dBd.

If the enzyme gain was 10× then E=−20 dBd. The 2D secondary array wouldthen become: −14, −17, −20, −23, −26, −29, blank, blank dBd. The twodots towards 0% having been damaged enough by the antigen retrievalprocess that they are unrecoverable by the staining and thus, blank. Iffor example the 2D/3D color density difference is 10× then D=+20 dBdbringing the 3D secondary array to −34, −37, −40, −43, −46, −49, blank,blank dBd. It is assumed that the primary antibody reagents will findsuitable antigen sites in the primary targets that 100% yield takesplace. It is also assumed that while there are many more than twoantigen peptide strands per KLH protein that only one antibody caneffectively bind and become stained per KLH protein. Any additionalantibodies finding a suitable antigen on the same KLH protein will beprevented from completion by the secondary stain because of overlappingoccupancy. Therefore, the number of antigen sites per primary antigencarried protein that can become detected is one. Since the primarytargets contain the same number of proteins per micron as the secondarythe primary dilution from the 500 ug/ml antibody master is then appliedto the secondary array data to adjust the secondary color density tonumeric antigen density. Monitoring the secondary targets, choose thetarget that has a middle color density. The middle color density beingdefined as the 50% point between maximum black and maximum white. Thepoint then equates to 1.5 dBd out of the 3 dBd range. That point thenfunctions as the anchor upon which the antigen density ruler isestablished. Using the last target range above the midpoint becomes−41.5 dBd.

The secondary targets are diluted to a 10 μg/mL master dilution. Eacharray is a blend of mouse or rabbit mixed with donkey IgG proteins.While the proteins all have different atomic mass's the following willassume all are 150 kDa and that the total number of proteins per targetdot is constant the mix ratio is not. For now, only the reactive proteinconcentration is being considered. At 150 kDa the individual proteinmolecular weight MW=249.07×10⁻¹²ng. The standard target dot is 1 mm indiameter. If the printed deposit is 1 μm thick, and the depositconcentration is 10 μg/mL, 31.5×10⁶ proteins will be deposited. A 1 μmdiameter area would then have 31.5 proteins. If we allow that oneprotein equates to 1 antigen site then the antigen density can beestablished. The secondary array uses the same number of proteins perdeposit, but the ratio between mouse or rabbit and donkey changes as theconcentration of the Mouse or Rabbit is reduced. The 100% target isentirely Mouse or Rabbit and is matched to the 0 dBd point on the ruler.

The secondary staining kit will only stain on the tissue when a primaryantibody binds to an antigen site on the tissue. The concentration ofthe applied antibody is not particularly limited, except that asufficient antibody concentration is provided to bind to the availableantigen sites. Thus, the antigen density measurement on the tissueremains a constant, but the numeric values is corrected for antigenretrieval damage and secondary enzyme gain. The color density vs.numeric measurement is then harmonized.

In the previous example the enzyme gain is 10× and the antigen retrievalhas caused the loss of two dots from the secondary array. The enzymegain is −20 dBd while the antigen retrieval loss is +6 dBd. The resultis −14 dBd. The dilutions then translate to:

% Target Antigen Density Numeric Density Color Density Mouse/Rabbit:Mouse/Rabbit in dBd dBd Donkey 1 μm² 0 −14 100 31.5 −3 −17 70.8 23.32 −6−20 50.1 15.78 −9 −23 35.5 11.18 −12 −26 25.1 7.90 −15 −29 17.78 5.60−17 −32 12.59 3.96 −20 −35 9.9 3.12

Type B: Primary Antigen Based Ruler

This form uses both the primary and secondary target arrays. The passedin information that is imbedded in the 2D bar code includes the (a)primary antibody data: host species for the antibody and dilution in dBdand (b) secondary enzyme gain. The lot code data includes theinformation about which primary target combination is in use.

If a primary target series is present it would be 3-dots wherein themost concentrated dot would be at the same 100% concentration as thesecondary array, but the dots are spaced apart in −6 dBd steps. Ineffect, the primary array and secondary array have the same dilutionslope. The primary targets become: −0, −6, −12 dBd and are representedas PdBd (primary dB dilution). It is reasonable to expect that theantigen retrieval will damage will nearly identical to that of thesecondary array. The primary array is acted upon by the secondary stainand thus experiences the same enzyme gain function. Thus, the primaryarray would be (−A+AR−E)dBd, where the primary target density iscontrolled by the primary antibody dilution. The only requirement isthat P is always greater than A. For 10× enzyme gain=−20 dBd and +6 dbdantigen retrieval loss the primary array is −20, −26, −32 dBd. Theantigen retrieval loss does not act upon the primary targets enough toblank them out, based on the impact to the secondary array. While thesecondary array is sufficient to produce the antigen density rulers itis important to verify that the primary dilution was correctly applied.Thus, the primary targets function in that capacity.

The foregoing outlines features of several embodiments so that thoseskilled in the art may better understand the aspects of the presentdisclosure. Those skilled in the art should appreciate that they mayreadily use the present disclosure as a basis for designing or modifyingother processes and structures for carrying out the same purposes and/orachieving the same advantages of the embodiments introduced herein.Those skilled in the art should also realize that such equivalentconstructions do not depart from the spirit and scope of the presentdisclosure, and that they may make various changes, substitutions, andalterations herein without departing from the spirit and scope of thepresent disclosure.

What is claimed is:
 1. A slide, comprising: a detection area configuredto hold a sample comprising a tissue section or loose cells; and acontrol area configured to: indicate an error and performance measure ofintermediate steps during an immunohistochemical or an immunochemicaldetection process, and provide a reference for qualitatively orquantitatively determining a color and antigen density of the stainedtissue or cells.
 2. The slide of claim 1, further comprises an adhesivecoating configured to bind with moieties, wherein the adhesive coatingcomprises at least one end group selected from the group consisting of—ROH, —R(C═O)OH, —RNH3, —R(C═O)NH₂, and −RNH₂.
 3. The slide of claim 1,wherein the control area comprises a primary target array, the primarytarget array comprises one or more primary target loading dots, eachprimary target loading dot of the one or more primary target loadingdots comprises a protein site including a protein reactive to a hostFcyRI peptide on the primary antibody, the protein is reactive to asingular or plurality of primary antibody hosts, and each protein siteimmobilizes a single primary antibody protein onto the slide.
 4. Theslide of claim 3, wherein the FcyRI peptide is coupled to a carrierprotein, and the carrier protein comprises a non-reactive protein, thenon-reactive protein is non-reactive to a secondary stain reagent usedin the immunohistochemical or the immunochemical detection process. 5.The slide of claim 4, wherein the primary target array comprises aplurality of primary target loading dots, each primary target loadingdot of the plurality of primary target loading dots comprises a samereactive FcyRI peptide, each primary target loading dot of the pluralityof primary target loading dots has a different concentration of thereactive FcyRI peptide from each other primary target loading dot of theplurality of primary target loading dots.
 6. The slide of claim 4,wherein the primary target array comprises a plurality of primary targetloading dots, each primary target loading dot of the plurality ofprimary target loading dots comprises a different reactive peptide, eachprimary target loading dot of the plurality of primary target loadingdots has a different concentration of the reactive peptide from eachother primary target loading dot of the plurality of primary targetloading dots.
 7. The slide of claim 1, wherein the control areacomprises a primary target array, the primary target array comprises aprimary target loading dot, the primary target loading dot comprises aplurality of primary targets, and each of the plurality of primarytarget comprises an antibody against a host protein of the primaryantibody used in the immunohistochemical or the immunochemical detectionprocess.
 8. The slide of claim 1, wherein the control area comprises aprimary target array, the primary target array comprises a primaryloading dot, the primary target loading dot comprises a plurality ofprimary targets, and each of the plurality of primary target comprisesProtein A, Protein G, Protein A/G, or Protein L against the host proteinof the primary antibody used in the immunohistochemical or theimmunochemical detection process.
 9. The slide of claim 1, wherein thecontrol area comprises a secondary target array, the secondary targetarray comprises a secondary target loading dot, the secondary targetloading dot comprises a plurality of secondary targets, and each of theplurality of secondary targets comprises: a host protein of a primaryantibody used in the immunohistochemical or the immunochemical detectionprocess; and a dummy protein.
 10. The slide of claim 9, wherein thecontrol area comprises a plurality of secondary target arrays, each ofthe plurality of secondary target arrays in a same secondary targetarray comprises a same host protein, and each of the plurality ofsecondary target arrays in different secondary target arrays comprisesdifferent host proteins.
 11. The slide of claim 1, wherein the controlarea further comprises an imaging reference loading dot.
 12. The slideas claimed in claim 11, wherein the imaging reference loading dotcomprises at least one: a black reference target comprising carbon dust;a white reference target, wherein the white reference target comprises ametal oxide or a metal sulfate; or a clear reference target, wherein theclear reference target comprises a non-reactive protein to anyimmunohistochemical or immunochemical reagents.
 13. The slide of claim11, wherein the imaging reference loading dot comprises: ananhydride-based epoxy for a white reference target and a black referencetarget; and a protein from the equine family of mammals for a clearreference target.
 14. The slide of claim 1, wherein the control areafurther comprises an antigen retrieval monitor loading dot configured tomonitor a degree of antigen retrieval in the immunohistochemical or theimmunochemical detection process.
 15. The slide of claim 14, wherein theantigen retrieval monitor loading dot comprises a carrier protein orsubmicron bead comprising: host proteins covalently attached fordefining a 3D particle; and a fixative under or over the host proteins.16. The slide of claim 1, wherein the control area is covered with aparaffin coating.
 17. The slide of claims 1, further comprises at leastone of: a marking identifying a slide type; a code for identifying anantigen supported by the control area; or a lot number.
 18. A method forimmunohistochemical staining using a slide, the method comprising:removing a portion of a fixation, wherein the portion is sufficient toexpose antigen sites in a tissue section and expose control targets;staining the tissue section and the control targets, wherein thestaining comprising: applying one or more primary antibodies to theslide, and applying one or more secondary antibodies conjugated with amoiety to the slide; or applying one or more primary antibodiesconjugated with the moiety to the slide; and applying the stain reagentsthat produce color in a presence of the moiety to indicate a presence ofa targeted antigen; assessing a quality of the staining process, whereinthe assessing comprises assessing staining results of the controltargets or an antigen retrieval monitor located in a control areaseparate from the tissue section.
 19. The method of claim 18, furthercomprising removing a paraffin protection layer from the controltargets, wherein removing the paraffin protection layer comprises:warming the slide to a temperature ranging between 65 and 75° C. for3-10 minutes to obtain a semi-liquefied paraffin over the control areaand tissue section; liquefying the semi-liquefied paraffin with anorganic solvent; rehydrating the control and detection areas using astepped sequence of anhydrous ethanol to at most 60% ethanolconcentration; and rehydrating the control targets in a buffer solution.20. The method of claim 19, wherein the organic solvent comprises atleast one selected from the group consisting of an aliphatic solvent,xylene, and xylol, and the buffer solution is a salt-based buffersolution.